Isobaric mass tagging or labeling is a method for determining the relative abundance of a given peptide across multiple samples by mass spectrometry. Each sample, typically representing a distinct biological condition, is reacted with a different isotopic variant of a labeling reagent. The samples are mixed together and analyzed by MS/MS. When a given peptide (isobaric molecules collected from all conditions) is isolated and fragmented, reporter ions arising from the various labeling reagents have different nominal masses, identifying the origin of each peptide, and allowing relative quantification of the peptide across samples. Sets of isobaric mass tagging reagents are commercially available from Thermo Fisher Scientific Inc, under the trade name Tandem Mass Tags (TMT), and from AB Sciex under the trade name Isobaric Tags for Relative and Absolute Quantitation (iTRAQ). The structure and utilization of isobaric mass tags has been described extensively in the patent prior art (see, for example, U.S. Pat. No. 7,816,304 by Schmidt et al. and U.S. Pat. No. 7,732,378 by Thompson et al., the disclosures of which are both incorporated herein by reference) as well as in the scientific literature (see, for example, Ross et al., “Multiplexed Protein Quantitation in Saccharomyces cerevisiaie Using Amine-reactive Isobaric Tagging Reagents”, Molecular & Cellular Proteomics, 3(12), pp. 1154-1167 (2004); and, Dayon et al., “Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS/MS Using 6-Plex Isobaric Tags”, Analytical Chemistry, Vol. 80, pp. 2921-2931 (2008)).
Considerable effort has been invested in increasing the “multiplex” capacity of the isobaric mass tag system. For example, the “sixplex TMT” reagent set has additional value over the “fourplex iTRAQ” reagent set because it allows the user to compare more samples with fewer chromatographic runs. The multiplex capacity of the system is ultimately limited by the number of atoms in the reporter region of the isobaric reagent available for isotopic substitution and the difficulty and cost associated with introducing a large number of isotopic substitutions.
U.S. Patent Application Publication No. 2010/0029495 to Schaefer describes an approach to expanding multiplexing capacity by providing a set of labeling reagents which includes a plurality of reagents having reporter groups of the same mass but of different molecular structures. Its technique involves performing a second stage of fragmentation (MS3) to cause fragmentation of the reporter ions, such that each same-mass reporter group yields characteristic product ions that may be distinguished in the mass spectrum, thereby permitting assignment of the relevant intensity to the corresponding labeled sample. While this approach may provide for a greater degree of multiplexing, the requirement of performing MS3 to distinguish different reporter ions of the same mass complicates the analysis, and may reduce sensitivity and throughput.